Summary The overall goal of our study is to identify small molecule inhibitors with appropriate potency, selectivity, and safety profiles to cure chronic toxoplasmosis. Toxoplasma gondii is a widespread parasite of animals that causes opportunistic infections in humans. Although healthy individuals control the infection, they are not able to completely eliminate it and remain chronically infected. Complications occur due to reactivation of chronic infections in immunocompromised patients and new infections during pregnancy when the parasite can cross the placental barrier and infect the developing fetus. It is estimated that ~ 2 billion people worldwide are chronically infected with T. gondii and hence at risk of reactivation should their immune function decline. Existing chemotherapy for T. gondii is only effective at suppressing acute infection, but is unable to eradicate the chronic tissue-cyst stages. In preliminary studies, we have conducted a high throughput screen of a diverse small molecule library of compounds and identified a number of very potent inhibitors (low nanomolar EC50) of parasite growth in vitro. Additionally, several of these highly potent leads act on chronic stages of infection and suppress reactivation of infection in an immunocompromised mouse model of toxoplasmosis. The active compounds come from a diversity-oriented synthetic (DOS) library that was synthesized using modern methods of asymmetric organic chemistry, hence they are rich in stereochemical diversity. The proposed studies will focus on two series that display low nM potency in inhibiting T. gondii growth. We will develop structure activity relationships based on T. gondii growth inhibition by existing analogs. We will then design and synthesize new analogs to optimize potency, safety, metabolic, brain penetration, and pharmacokinetic properties. Genetic and genomic approaches will be used to identify potential resistance and confirm mechanisms of action of these leads. We will also employ animal models that we have developed for monitoring the efficacy of compounds against reactivated toxoplasmosis. Successful achievement of these goals will define leads for future clinical studies aimed at developing new drugs to eradicate chronic toxoplasmosis.